Genomic sequencing has shown that the CRP/CooA superfamily of proteins is quite large and must regulate a variety of disparate functions. However, only a modest number of members of this superfamily are understood in terms of their effectors and their physiological role. We have had great success in analyzing the response of CooA to CO binding and the basis of its specificity for CO. We have already extended some of those insights to provide a better understanding of the response of CRP itself to its effector, cAMP. Nevertheless, some key issues remain that we will address in Aim I of this proposal, including (i) the basis for repositioning of the DNA-binding domain in response to effector;(ii) the degree to which the each protein's ligand specificity is indirectly altered by properties physically unlinked to the effector-binding site;and (iii) the identification of the structures for each protein that are necessary for a full understanding of their responses to effector. In another aim of the proposal, we will exploit our understanding of CRP and CooA to develop novel and general methods for understanding the effectors and the physiological roles of other members of the superfamily that have been identified through genome sequencing. In the course of our work on CooA, we have found a completely different CO-sensing transcriptional factor in R. rubrum and other bacterial species, which we have termed RcoM (regulator of CO metabolism). By a variety of criteria RcoM appears to be a CO-sensor that regulates the expression of aerobic CO dehydrogenase systems. The RcoM family contains heme, but is not homologous to CooA. Its effector-binding domain is homologous to that of other heme-containing PAS domains and its DNA-binding domain, termed a LytTR domain, is distributed broadly in prokaryotes, yet its function is poorly understood. We have already developed purification methods, an in vitro functional assay and identified one heme ligand in RcoM. We will exploit these tools and the methodology we developed for CooA to analyze CO responsiveness in this novel family of CO sensors.